PROJECT SUMMARY Lifespan, disease susceptibility, and general wellness are phenotypes that vary substantially among individuals. These traits show variation even when individuals are isogenic such as in twins or model organisms. The molecular determinants behind this variation have long been sought, along with the ability to predict how one individual will differ from another. In the nematode Caenorhabditis elegans, variation in lifespan and wellness traits among isogenic individuals has recently been linked to differences in expression of molecular chaperones. These expression differences are heritable, implying a stable epigenetic or unstable genetic source. We propose copy number variation in ribosomal RNA genes as an underlying genetic cause of phenotypic variation. Ribosomal RNA genes (rDNA) exist as tandem gene arrays in all eukaryotes. Their repetitive nature predisposes them to instability and thus copy number variation among individuals, making them heritable yet highly mutable genomic elements. Our preliminary data demonstrate that vast differences in rDNA copy number exist among C. elegans strains isolated from the wild, as well as among laboratory worms only ten generations removed from a common ancestor. rDNA dosage is known to affect global gene expression in flies and humans, and genome integrity and replicative lifespan in yeast. Furthermore, the nucleolus itself, which is comprised of rDNA, is a major hub of stress response and protein homeostasis. Despite its extraordinary importance, rDNA and its copy number variation are grievously understudied, due largely to the inherent technical challenges involved in genotyping repetitive DNA. In this proposal, we will develop a new, universally-applicable technology to genotype rDNA copy number across many individuals, and explore the phenotypic consequences of rDNA copy number variation. In Aim 1, we will leverage single- molecule-enabled sequence capture to accurately call rDNA copy number in wild isolates and laboratory strains of C. elegans, at both the population and individual levels. This high-throughput technology will be immediately applicable for future rDNA genotype-phenotype association studies in humans. Facilitated by our new genotyping method, in Aim 2, we will test correlations between rDNA copy number and candidate phenotypes, including longevity, stress responses, mutation penetrance, motility, proteostasis, development, and fecundity across genetically diverse wild isolates and nominally isogenic C. elegans laboratory strains. To test causality of rDNA genotype for phenotype, we will use the CRISPR-Cas system to manipulate rDNA copy number, among other approaches, and characterize the phenotypic consequences for lifespan and wellness. In Aim 3, we will evaluate potential molecular mechanisms through which rDNA dosage may affect phenotype in C. elegans. We will determine if rDNA copy number variation affects rRNA expression, replication stress, extrachromosomal circular rDNA abundance, or mutagen sensitivity. Taken together, this proposal explores a currently uncharacterized class of genetic variation with potentially vast phenotypic impact and develops technology that makes this genetic variation accessible for future studies of aging and disease susceptibility in humans.